1. Field of the Invention
This invention relates generally to the fields of Raman spectroscopy and imaging. Specifically, the present invention relates to a nano-imaging probe useful in surface enhanced Raman spectroscopic applications.
2. Description of the Related Art
Recent technological advances in the controlled fabrication and manipulation of nanoscale systems and devices has generated increased interest in various nano-structures, both man-made, e.g., carbon nanotubes, (1-2) and naturally occurring, that is, biological cells or subcellular compartments (3). Synthesizing, characterizing and understanding such structures and their function, therefore, is extremely important to many fields ranging from drug delivery in the design of effective time-release materials (4-5) to space exploration in the fabrication of light-weight, rugged materials (6). In order to better characterize and further develop these nano-structures, the ability to visualize objects on the nanometer to hundreds of nanometer's scale is essential.
One area in which high-resolution imaging of individual chemical species will have a great impact is in biochemical studies. For example, a novel tool for characterizing dynamic intracellular and extracellular interactions on the nanoscale could offer insight into metabolic pathways, ion exchange mechanisms, and other essential cellular processes. In medical research, a tool for measuring and imaging localized chemical changes at the cellular and sub-cellular levels could allow for pre-symptomatic disease detection as well as potentially allow for the development of specific treatments for individuals (7-10).
Chemical imaging using optical spectroscopies has long been used for the identification and the determination of the spatial distribution of species within a sample. Using different optical spectroscopic techniques, i.e., fluorescence (11-13), Raman (14-18) and others (19-20), it is possible to obtain both qualitative, as well as quantitative, information about a sample. Fluorescence imaging is commonly used for the monitoring of trace amounts of a known analyte and Raman imaging is typically used for species identification. Recently, surface enhanced Raman spectroscopy (SERS) also has been employed for chemical imaging, providing both the qualitative identification of Raman imaging with the sensitivity necessary for trace analyses (21). However, the creation of uniform roughened metal surfaces, necessary for SERS, and thus uniform surface enhancement, is difficult to control from location-to-location which dramatically limits the use of SERS as an imaging technique (22-25). Additionally, the far-field imaging methodologies employed in conventional chemical imaging analyses ultimately limits the spatial resolution of such analyses to the diffraction limit of light, effectively preventing the imaging of nanometer scale objects.
To overcome this diffraction limited spatial resolution and to obtain chemical images with resolution on the nanometer scale, near-field chemical imaging techniques were developed, including near-field scanning optical microscopy (NSOM) (26-29). Coupling NSOM with the sensitivity of fluorescence spectroscopy, near-field chemical images of samples have been obtained for many different applications, including medical diagnostics (30), materials science studies (27,31) computer science (32) and chemical and biological research (33). More recently, the nanometer scale fiber optic tip used for scanning in most NSOM analyses was scanned over a SERS active surface to record SERS images with approximately 100 nm spatial resolution. This has resulted in the ability to visualize individual molecules, such as a single strand of DNA labeled with brilliant cresyl blue (BCB) (34-35).
While NSOM in conjunction with other spectroscopic techniques can be used to produce high-resolution chemical images below the diffraction limit of light, dynamic real-time sample imaging has not yet been feasible, as NSOM requires hours of scanning the probe tip across the surface (36). In addition, if a particular area of interest is found, it is often difficult to return to this exact location after the scan has been completed (36). Due to these limitations, NSOM is not particularly suited to studying dynamic chemical events.
Accordingly, a need is recognized in the art for improved surface enhanced Raman spectroscopic imagers useful for real time, reproducible imaging in a non-scanning format to overcome the challenge of SERS substrate reproducibility in SERS imaging and the dynamic limitation of NSOM. More specifically, the prior art is deficient in a SERS nanoimaging probe having a uniform, repeatable and regular nano-structured surface effective for nanoscale resolution and methods of making the same. The present invention fulfills this longstanding need and desire in the art.